Changes in Transcript Abundance Relating to Colony Collapse Disorder in Honey Bees (Apis Mellifera)

Changes in transcript abundance relating to colony collapse disorder in honey bees (Apis mellifera)

Reed M. Johnsona,1, Jay D. Evansb, Gene E. Robinsona, and May R. Berenbauma,2

aDepartment of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL 61801; and bBee Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705

Contributed by May R. Berenbaum, July 14, 2009 (sent for review February 18, 2009) Colony collapse disorder (CCD) is a mysterious disappearance of Pesticides also have been suspected to play a role in CCD. honey bees that has beset beekeepers in the United States since Sublethal exposures leading to behavioral disruptions consistent late 2006. Pathogens and other environmental stresses, including with the failure of foragers to return to their hives have been pesticides, have been linked to CCD, but a causal relationship has associated with the neonicotinoid pesticides (7). The long-term not yet been demonstrated. Because the gut acts as a primary use of combinations of in-hive pesticides for control of honey bee interface between the honey bee and its environment as a site of parasites also may have contributed to otherwise unexplained entry for pathogens and toxins, we used whole-genome microar- bee mortality (8). rays to compare gene expression between guts of bees from CCD To differentiate among possible explanations for CCD, we colonies originating on both the east and west coasts of the United used whole-genome microarray analysis, comparing gut gene States and guts of bees from healthy colonies sampled before the expression in adult worker bees from healthy and CCD colonies. emergence of CCD. Considerable variation in gene expression was We assayed the gut because it is the principal site of pesticide associated with the geographical origin of bees, but a consensus detoxification and an integral component in the immune defense list of 65 transcripts was identified as potential markers for CCD against pathogens in A. mellifera. Samples from colonies varying status. Overall, elevated expression of pesticide response genes in CCD severity were collected on the U.S. east and west coasts was not observed. Genes involved in immune response showed no in the winter of 2006–2007 and compared with healthy (‘‘his- clear trend in expression pattern despite the increased prevalence torical’’) controls collected in 2004 and 2005. of viruses and other pathogens in CCD colonies. Microarray analysis revealed unusual ribosomal RNA fragments that were con- Results spicuously more abundant in the guts of CCD bees. The presence Gene Expression Differences. The microarray contained oligonu- of these fragments may be a possible consequence of picorna-like cleotide probes representing 9,867 different genes (with dupli- viral infection, including deformed wing virus and Israeli acute cate spots), based on gene predictions and annotation from the paralysis virus, and may be related to arrested translation. Ribo- honey bee genome sequencing project; in addition, 2,729 probes somal fragment abundance and presence of multiple viruses may specific for ESTs matching no existing annotation were repre- prove to be useful diagnostic markers for colonies afflicted with sented on the array (9–11), including 21 probes derived from CCD. expressed sequence tags (ESTs) specific for rRNA [supporting information (SI) Table S1]. microarray ͉ picorna-like virus ͉ ribosomal RNA Of the 6,777 probes with expression above background level, 1,305 probes showed significant differences in expression in at s the premier managed pollinator in the United States, the least 1 of the 5 contrasts [false discovery rate (FDR), P Ͻ .01; Awestern honey bee, Apis mellifera, contributes more than and fold-change Ͼ2) (Fig. 1]. The west coast versus historical $14 billion to agriculture annually (1). Beginning in fall 2006, the comparison alone generated a list of 948 differentially expressed American apiculture industry experienced catastrophic losses of probes; however, 668 of these were not differentially expressed unknown origin. The phenomenon, called colony collapse dis- in any of the other comparisons. order (CCD), was identified by a set of distinctive characteristics, There was little concordance in the lists of differentially including the absence of dead bees in or near the colony and the expressed genes between the east and west coast comparison of presence of abundant brood, honey, and pollen despite vastly guts taken from ‘‘severe’’ and ‘‘mild’’ colonies. Just 54 probes reduced numbers of adult workers (2). Losses were estimated at shared differential expression between the east and west coast 23% over the winter of 2006–2007 (3) and at 36% over the winter severe versus mild comparisons, with 191 probes showing unique of 2007–2008 (4). differential expression. That so many genes are differentially tran- Using metagenomics, Cox-Foster et al. (3) compared the scribed in the severe versus mild comparisons suggests these microbial flora of honey bees in hives diagnosed with CCD and classifications may not be equivalent across geographical samplings. in ostensibly healthy hives, evaluating pathogens with respect to Reflecting the strong differences in gene expression, hierar- their association with diagnosed CCD. The most predictive chical cluster analysis, using expression values for all probes, pathogen was Israeli acute paralysis virus (IAPV), a picorna-like virus (Dicistroviridae) hitherto unreported in the United States. However, a later study found IAPV in U.S. bees before the Author contributions: R.M.J., J.D.E., G.E.R., and M.R.B. designed research; R.M.J. and J.D.E. appearance of CCD (5), discounting an exclusive causal rela- performed research; J.D.E. and G.E.R. contributed new reagents/analytic tools; R.M.J., J.D.E., G.E.R., and M.R.B. analyzed data; and R.M.J., J.D.E., G.E.R., and M.R.B. wrote the tionship. The metagenomic analysis identified other pathogens paper. associated with CCD bees, including the picorna-like viruses The authors declare no conflict of interest. Kashmir bee virus (KBV) and deformed wing virus (DWV), and Data deposition: The data reported in this paper have been deposited in the ArrayExpress 2 species in the microsporidian genus Nosema (3), one of which, database, www.ebi.ac.uk/arrayexpress (accession no. E-MEXP-2292). N. ceranae, was subsequently linked to collapses of colonies in 1Present address: Department of Entomology, University of Nebraska-Lincoln, Lincoln, NE Spain (6), although this may be unrelated to CCD in the United 68583. States. A high prevalence of multiple pathogens in CCD bees 2To whom correspondence should be addressed. E-mail: [email protected]. suggests that a compromised immune response may be integral This article contains supporting information online at www.pnas.org/cgi/content/full/ to CCD. 0906970106/DCSupplemental.

14790–14795 ͉ PNAS ͉ September 1, 2009 ͉ vol. 106 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906970106 Downloaded by guest on September 26, 2021 catabolic process (GO:0009253) were overrepresented but did not demonstrate consistent directional differences. The peptidoglycan recognition proteins (PGRPs) contribute to immunity by detecting bacteria (13); PGRP-S2 was up-regulated and PGRP-S3 was down-regulated in CCD bees. Transcripts encod- ing sallimus, a large complex protein that mediates muscle elasticity (14), were more abundant in west coast bees. This protein also may contribute to immunity, because it contains Ig repeats and its transcripts are overexpressed in Anopheles gam- biae mosquitoes after bacterial infection (15). The gene mblk-1 was more highly expressed in CCD bees; the D. melanogaster ortholog of this gene plays a role in hormone- triggered cell death (16) and larval midgut histolysis (17). Thus, overexpression of mblk-1 may indicate apoptosis in the guts of CCD bees. However, of the 39 apoptosis-related (GO:0006915) genes on the microarray, only mblk-1 and GB14659, which is similar to apoptotic peptidase activating factor 1, were more highly Fig. 1. Experimental design for microarray comparisons of CCD and healthy expressed in the guts of CCD bees. bees. East coast CCD includes bees collected in Florida and Pennsylvania, and west coast CCD includes bees from California. Guts from bees remaining in colonies that were classiﬁed as mild or severe were compared with each other Pathogens in CCD Bees. Twenty-two probes specific for 8 bee and together, through a reference gut RNA sample, with guts of healthy pathogens were present on the microarray [KBV, DWV, black historical bees collected in 2004 and 2005. A total of 22 microarrays were queen cell virus (BQCV), acute bee paralysis virus, sacbrood hybridized. virus, Ascophaera apis, Nosema apis, and Paenibacillus larvae] (Fig. S2), providing an opportunity to survey the samples for pathogens. In some cases, the number of colonies sampled was grouped the samples broadly by health status and geography (Fig. small with respect to standard pathogen surveys, so the results 2). All healthy bees clustered together, as did all CCD bees, which are best interpreted in a qualitative rather than quantitative were further clustered according to geography, with west coast manner. Three pathogens were detectable at different levels California bees forming one group and east coast Pennsylvania and among the sampled bees. Chalkbrood (A. apis) RNA was more Florida bees clustering together in another group. Differences in abundant in both east coast CCD bees and in severe CCD bees. relative colony health at the time of collection, either mild or severe BQCV RNA was more abundant in CCD bees, as was DWV, but CCD, were not reflected in the cluster analysis. DWV also was more abundant in east coast severe CCD bees. Although geographic variation among the east and west coast The DWV probe on the microarray also may have detected the CCD samples is evident, if CCD is a distinct phenomenon, then closely related Kakugo virus or Varroa destructor virus 1 (18), it should be possible to identify a list of CCD-related genes that with which it shares 91% identity. A probe specific for IAPV (19) show differential expression in both east and west coast CCD was not included on the array and likely is not detected by the bees relative to healthy bees. But because these comparisons rely probe for KBV, with which it shares just 60% identity. The on the historical samples, which may carry their own geographic presence of Nosema, IAPV, and other viruses was surveyed with biases, we further winnowed the gene list by using only genes quantitative PCR (qPCR), indicating a generally higher viral differentially expressed in the east coast severe versus mild load in CCD colonies (Table 3). comparison (Fig. 3). This comparison was informative; expression of 65 transcripts was consistently up or down when com- Validation of Microarray Results with qPCR. Expression changes of paring relatively sick bees (CCD or severe) and relatively healthy 8 transcripts were verified by qPCR. Five of 8 transcripts had bees (historically healthy or mild) (Fig. 2); 29 probes on this list similar expression profiles when measured using the same RNA correspond to annotated genes, 35 probes correspond to ESTs, samples analyzed by both methods (Table S4). Biological vali- and 1 probe corresponds to a pathogen, DWV. dation of microarray results was performed using qPCR on gut samples from healthy and CCD bees collected in California in Gene Ontology Functional Analyses of Gene Expression Differences. 2007, a year after the samples used for microarray analysis were Genes differentially expressed between groups were categorized obtained. Differential expression of 3 of 8 transcripts was according to Gene Ontology (GO) (12). FlyBase orthologs of confirmed: CCD-associated up-regulation of the ESTs QW33 bee genes that were differentially expressed were used for GO and jdeC15, corresponding to 28S and 5.8S rRNA subunits. enrichment analyses (Table 1). In the cellular component on- DWV also was more abundant in CCD gut samples (Table 2). tology, genes associated with lipid particle (GO:0005811), cyto- Further qPCR validation of markers was performed with solic small ribosomal subunit (GO:0022627), and mitochondrial abdomens of bees from 147 colonies in healthy and CCD apiaries respiratory chain (GO:0005746) generally demonstrated re- in 6 states. Only an rRNA-coding EST, QW33, was differentially duced expression in west coast bee guts relative to historical expressed between CCD and healthy abdomens (Table 2). These samples. CCD bees also were more likely to harbor both a larger number In the molecular function ontology, genes related to transcrip- of viruses (as detected by these methods) and nonviral patho- SCIENCES

tion factor activity (GO:0003700) were more highly expressed in gens, as in the original samples (Table 3). AGRICULTURAL both east coast and west coast CCD bees and in severe CCD bees. West coast bees also showed reduced expression of both Discussion heme binding (GO:0020037) and carboxylesterase activity No simple explanations for the cause of CCD emerge from the (GO:0004091) categorizations, which include cytochrome P450 microarray analysis. Expression of detoxification and immune monooxygenases (P450s) and carboxylesterases (COEs), the gene transcripts, which would indicate toxins or disease as the principal xenobiotic metabolizing enzymes. One P450, cause of CCD, was largely unchanged. However, considerable CYP4G11, was expressed at a lower level in all 3 contrasts tested. geographic variation existed among CCD bees, with west coast In the biological process ontology, genes related to develop- bees seemingly more severely affected. Transcripts of genes ment (GO:0016348 and GO:0007509) and the peptidoglycan related to basic cellular processes involving ribosomal and

Johnson et al. PNAS ͉ September 1, 2009 ͉ vol. 106 ͉ no. 35 ͉ 14791 Downloaded by guest on September 26, 2021 Fig. 2. Heatmap showing expression values for 65 probes demonstrating differential expression in 3 contrasts: east coast CCD versus historical, west coast CCD versus historical, and east coast severe versus east coast mild [P Ͻ .01 (FDR); fold-change Ͼ2]. Yellow indicates increased transcript abundance and red indicates de- creased transcript abundance relative to the mean for all colonies. Probes lacking a description correspond to EST sequences for which no matching gene has been found. More information about the probes is provided in Table S5. CA, California; PA, Pennsylvania; FL, Flor- ida. Numbers correspond to colony number. S, severe CCD; M, mild CCD. Colonywise clustering was performed using expression values for all 6,777 probes. Euclidean distances were calculated and clustered using the ‘‘complete’’ method.

mitochondrial function were generally less abundant in west CYP4G11 was elevated in CCD bees, but the function of this coast bees. P450 remains uncharacterized. Its ortholog in D. melanogaster, P450s and COEs play multiple metabolic roles and are par- CYP4G15, is expressed only in the central nervous system, where ticularly important in the detoxification of natural and synthetic it may be associated with ecdysteroid metabolism (21). The toxins in bees and other insects (20). However, the few genes in single COE overexpressed in CCD bees, GB10854, differs from these superfamilies differentially transcribed with CCD likely other insect COEs in terms of its catalytic site, such that it may serve functions other than detoxification. Expression of be incapable of detoxificative carboxylester hydrolysis (22).

14792 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906970106 Johnson et al. Downloaded by guest on September 26, 2021 rently defies technical explanation; differential expression of these transcripts associated with age and caste in a termite (Reticulotermes flavipes) EST project (28) suggests an underlying biological explanation. One possible explanation for the presence of poly(A) rRNA sequences in bees is that they are degradation intermediates. Polyadenylation is a well-known marker of RNA degradation in bacteria and organelles (29), and poly(A) addition is known to contribute to eukaryotic rRNA degradation (30). Polyadenyl- ated 25S rRNA comprises Ϸ0.02% of all of the 25S rRNA transcripts in fission yeast Schizosaccharomyces pombe (31). Another explanation for these sequences is the ‘‘ribosomal filter hypothesis,’’ in which conventional mRNA transcripts include tens to hundreds of nucleotides similar to rRNA sequence in either the sense or antisense direction (32). Transcripts of genes containing rRNA sequence can bind to ribosomal subunits and alter translation efficiency (33). Short sections of rRNA function as an internal ribosome entry site (IRES) to interact with the ribosome and alter translation. Indeed, functions have been assigned to some mRNAs containing large sections of rRNA sequence (34), although none of the predicted genes annotated from the honey bee genome contains sufficient rRNA sequence Fig. 3. Venn diagram showing the number of transcripts up-regulated and to hybridize to any of the rRNA probes on the array (Table S1). down-regulated in 3 comparisons: west coast (California) CCD versus historical, east coast (Florida and Pennsylvania) CCD versus historical, and east coast A related hypothesis posits that long sequences of rRNA-like severe CCD versus mild CCD. transcript may aid in protein folding, taking on part of the enzymatic activity of rRNA (35). The picorna-like virus DWV was more abundant in CCD bees Although 88 known immune-related genes were included on analyzed in the microarray experiment and a broader sampling the microarray, these also were mostly unchanged in expression of bee colonies revealed that CCD bees carry a larger number in CCD bees. Given the association between pathogens and CCD of different picorna-like viruses (Table 3). Picorna-like viruses, (3), the finding that so few immune genes were differentially including IAPV and KBV, that have been associated with CCD expressed in CCD bees was unexpected. Insect guts can produce (3) initiate transcription of viral proteins by the ribosome antimicrobial peptides, including the apidaecins in honey bees through an IRES rather than through the 5Ј-methylation cap (23), yet CCD-related changes in transcription of these genes that initiates translation of most mRNA (36). The IRES se- were not detected. This lack of response does not rule out viral quences from both DWV and Varroa destructor virus 1 effectively infection, however, because antimicrobial proteins are ineffec- enhance translation of a reporter gene (36). Picorna-like viruses tive against viruses. In D. melanogaster, fungal or bacterial in mammals also halt translation of host mRNA through cleav- infection induces antimicrobial peptides, but flies infected with age of the translation initiation factor eIF4G and the poly(A)- Drosophila C virus (DCV), another picorna-like virus, did not binding protein (37), leaving the ribosomes incapable of binding produce more antimicrobial peptides (24) and demonstrated host mRNA and giving the viral RNA little competition for its elevated transcription of only a small number of immune genes IRES-mediated binding to host ribosomes (38). relative to fungal and bacterial infection (25). While there is no direct evidence that elevated poly(A) rRNA Many of the transcripts differentially expressed in CCD bees is a result of picorna-like viral infection, one consequence of the were detected by probes based on ESTs matching no existing viral disabling of ribosomal function may well be increased ribosomal degradation. Picornavirus infection in mammals both annotation (10, 11). Of the 2,729 geneless ESTs on the array, 28 reduces protein production and causes strings of translating were differentially expressed in CCD bees. Of all of the ESTs on polyribosomes to break down, and these idle ribosomal subunits which probes were based, 13 were discovered to contain rRNA may be more susceptible to degradation (39). Ribosomal deg- sequence, and 5 of these were more highly expressed in CCD bee radation also could occur in CCD bees independent of viral guts. These rRNA-coding ESTs were not identified as rRNA infection; for example, starving bacteria demonstrate elevated previously, because the A. mellifera rRNA sequence is not ribosomal degradation (39). Regardless of the cause, the abun- available in online repositories, and identification of these dance of poly(A) rRNA may be a useful diagnostic marker for probes was accomplished only by manually transcribing the determining CCD status, although the presence of poly(A) published rRNA sequence from printed text (26). Had these rRNA does not in itself indicate CCD, because healthy bees, ESTs been previously identified as rRNA, the associated probes including those used to generate EST libraries, contain these likely would have been excluded from the microarray. fragments. The presence of rRNA on these microarrays is surprising, Although gene transcript analysis did not clearly identify a because both the EST projects on which the probes were based specific cause for CCD, our study documents several patterns and the microarray hybridization rely on a polyadenylated 3Ј tail SCIENCES suggestive of a causal mechanism. The reduced protein synthetic AGRICULTURAL for the initial priming step in the reverse-transcription reaction. capabilities that would accompany ribosomal hijacking by mul- Ribosomal RNA is transcribed by RNA polymerase I, and the tiple picorna-like viruses would leave bees unable to respond to resulting transcript is not polyadenylated; as such, oligo(dT)- additional stresses from pesticides, nutrition, or pathogens. based reverse-transcription reactions should not reverse- Although any interpretation of the presence of these rRNA transcribe rRNA. Yet transcripts of rRNA have long appeared fragments is speculative, the reported interaction between bee in EST projects, and their reverse transcription has been attrib- picorna-like viruses and rRNA is suggestive of a possible root uted to poly(A)-rich internal sequences hybridizing to the oli- cause of CCD. go(dT) or to genomic DNA contamination (27). This differential To establish a causal relationship, the quantitative association expression of polyadenylated rRNA transcripts in insects cur- between multiple picorna-like virus infections and polyadenyl-

Johnson et al. PNAS ͉ September 1, 2009 ͉ vol. 106 ͉ no. 35 ͉ 14793 Downloaded by guest on September 26, 2021 Table 1. GO analyses for differentially expressed genes in 3 contrasts Change in Comparison GO ID Term Annotated Signiﬁcant Expected P value expression

Cellular component East vs. historical GO:0035102 PRC1 complex 3 2 0.09 .003 ϩ2/Ϫ0 GO:0005576 Extracellular region 124 11 3.6 .008 ϩ6/Ϫ5 West vs. historical GO:0005811 Lipid particle 205 46 26.72 Ͻ.001 ϩ6/Ϫ40 GO:0022627 Cytosolic small ribosomal subunit 30 10 3.91 .003 ϩ0/Ϫ10 GO:0005746 Mitochondrial respiratory chain 53 14 6.91 .006 ϩ4/Ϫ10 Molecular function East vs. historical GO:0003700 Transcription factor activity 144 15 3.91 Ͻ.001 ϩ14/Ϫ1 GO:0008599 Protein phosphatase type 1 reg. activity 2 2 0.05 .001 ϩ2/Ϫ0 GO:0043565 Sequence-speciﬁc DNA binding 75 8 2.04 .001 ϩ8/Ϫ0 GO:0008745 N-acetylmuramoyl-L-alanine amidase 3 2 0.08 .002 ϩ1/Ϫ1 GO:0005102 Receptor binding 41 6 1.11 .003 ϩ3/Ϫ3 GO:0005001 Transmembrane receptor protein tyrosine 4 2 0.11 .004 ϩ2/Ϫ0 phosphatase GO:0042834 Peptidoglycan binding 4 2 0.11 .004 ϩ1/Ϫ1 GO:0005372 Water transporter activity 5 2 0.14 .007 ϩ1/Ϫ1 West vs. historical GO:0043565 Sequence-speciﬁc DNA binding 75 18 9.2 .003 ϩ12/Ϫ6 GO:0003700 Transcription factor activity 144 29 17.66 .004 ϩ21/Ϫ8 GO:0004091 Carboxylesterase activity 26 9 3.19 .008 ϩ3/Ϫ6 GO:0020037 Heme binding 49 12 6.01 .013 ϩ1/Ϫ11 East mild vs. severe GO:0003700 Transcription factor activity 144 10 2.92 .001 ϩ4/Ϫ6 GO:0004558 Alpha-glucosidase activity 7 2 0.14 .008 ϩ2/Ϫ0 Biological process East vs. historical GO:0016348 Imaginal disc-derived leg joint 2 2 0.05 .001 ϩ2/Ϫ0 morphogenesis GO:0045449 Regulation of transcription 274 17 7.5 .001 ϩ16/Ϫ1 GO:0007415 Defasciculation of motor neuron axon 3 2 0.08 .002 ϩ2/Ϫ0 GO:0007509 Mesoderm migration 4 2 0.11 .004 ϩ2/Ϫ0 GO:0009253 Peptidoglycan catabolic process 4 2 0.11 .004 ϩ1/Ϫ1 GO:0035286 Leg segmentation 7 4 0.19 .007 ϩ4/Ϫ0 West vs. historical None East mild vs. severe GO:0006541 Glutamine metabolic process 4 2 0.09 .003 ϩ2/Ϫ0

GO terms signiﬁcantly enriched (P Ͻ 0.1, Fisher’s exact test) in the 3 gene lists are presented. The number of differentially expressed genes and the direction of change in expression relative to the more distressed second group in each comparison are listed for each GO term.

ated rRNA fragment abundance merits further exploration. In Our results also indicate an unappreciated variation in gene addition, the consequences of viral infection and CCD on the expression patterns and pathogen loads with geography. This function of ribosomes should be explored through assays of trans- variation provides insight into the different stresses facing bees lational efficiency. Because of the potential for translational inter- and clearly demonstrates that diagnostic surveys must sample ference, studies on immune suppression should focus on bioassays extensively across numerous bee populations. Colony surveil- or protein abundance rather than on immune gene transcripts. lance via assay of rRNA-like transcript abundance may provide

Table 2. Comparison of fold-change expression differences as measured by microarray and qPCR Fold-change microarray qPCR

Transcript name Entrez ID W vs. H E vs. H S vs. M Gut Abdomen

Deformed wing virus AJ489744.2 4.05 5.34 7.33 1.36* 3.00 Bee brain EST BI515001 1.21 0.95 0.68 — Ϫ1.21 mblk-1 NM࿝001011629.1 3.97 1.84 2.69 Ϫ1.51 — Cytochrome P450 (CYP4G11) NM࿝001040233.1 Ϫ4.01 Ϫ1.82 Ϫ7.44 Ϫ0.3 4.45 Peptidoglycan recognition prot. S3 XM࿝001123180.1 Ϫ1.94 Ϫ1.15 Ϫ2.47 Ϫ0.48 2.47 28S rRNA 401Ϫ469 (EST jdeC12) 4.67 3.58 3.94 0.72 — 5.8S rRNA 18Ϫ60 (EST jdeC15) 5.29 3.15 2.34 0.61* — 28S rRNA 1838Ϫ1903 (EST QW33) BG101565 2.92 2.80 3.99 1.92* 2.47*

Microarray: W vs. H, California 2006 CCD vs. historical; E vs. H, Florida and Pennsylvania CCD vs. historical; S vs. M, severe CCD vs mild CCD. QPCR: gut, California 2007 CCD vs. California 2007 healthy (n ϭ 60); abdomen, pooled abdomen samples collected from healthy and CCD bees in Pennsylvania, Florida, and California and healthy bees collected in Massachusetts and Illinois during 2006–2008 (n ϭ 147) (see Table S3). Transcripts showing similar trends in abundance in microarrays and qPCR samples (P Ͻ .05, one-tailed Mann-Whitney test) are indicated by * and using pooled abdomen samples collected from healthy and CCD bees in Pennsylvania, Florida, and California and healthy bees collected in Massachusetts and Illinois during 2006–2008 (n ϭ 147). *Signiﬁcance (P Ͻ .05).

14794 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906970106 Johnson et al. Downloaded by guest on September 26, 2021 Table 3. Proportion of colonies with detectable levels of viral and microsporidial pathogens, as measured by qPCR Number of Number of Year Status State Colonies ABPV KBV IAPV DWV SBV viruses N. apis N. ceranae Pathogens

2006 Healthy MA/PA 14 0% 14% 7% 64% 36% 1.21 Ϯ 0.80 0% 50% 1.71 Ϯ 0.99 CCD FL 24 38% 38% 25% 46% 8% 1.54 Ϯ 1.67 8% 42% 2.04 Ϯ 2.03 CCD CA 57 51% 21% 21% 58% 30% 1.81 Ϯ 1.41 40% 60% 2.81 Ϯ 1.74 2007 Healthy CA 14 86% 7% 14% 29% 7% 1.43 Ϯ 1.02 7% 86% 2.36 Ϯ 1.00 CCD CA 16 69% 44% 25% 44% 6% 1.88 Ϯ 0.72 0% 94% 2.81 Ϯ 0.83

Total number of different viruses and pathogens detected are summarized (Ϯ SD). More pathogens were detected in bees from colonies identiﬁed as suffering from CCD in both 2006 and 2007 (P Ͻ .05; one-way Mann-Whitney test), and CCD colonies overall contained more viruses (P Ͻ .05; Fisher’s combined probability test).

an earlier indication of CCD status than has hitherto been Dissection, RNA extraction, microarray, statistical and qPCR validation anal- available and allow beekeepers to take actions to reduce losses. yses are described in SI Text. Pooled samples of RNA from 6 guts collected from each CCD colony were hybridized to 2 arrays, incorporating a dye swap (Fig. Materials and Methods S1). Two separate pools of RNA from 6 guts each were created for both historical samplings, and each was hybridized against the reference on a single Biological Material and RNA Extraction. Remaining adult bees in colonies microarray. Experiments were designed to meet Minimum Information About diagnosed with CCD at apiaries in Florida, California, and Pennsylvania were a Microarray Experiment standards, and all microarray data obtained in these collected during winter 2006–2007. CCD colony health was scored at the time studies were deposited at ArrayExpress [www.ebi.ac.uk/arrayexpress (acces- of collection as either severe or mild, depending on the apparent strength of sion no. E-MEXP-2292)]. the colony. Historical bees, collected before the appearance of CCD and hence ostensibly healthy, were collected in 2004 and 2005 from colonies set up on new equipment with no miticide treatments in apiaries of Pennsylvania State ACKNOWLEDGMENTS. We thank D. vanEngelsdorp, J. Pettis, and D. Cox- University near State College, Pennsylvania (Table S3). Bees for the reference Foster for sample collection and colony health scores, and D. Lopez and T. Newman for laboratory assistance. We also thank I. Lipkin, J. Willis, and J. (ostensibly healthy) sample were collected from 5 hives near Urbana, Illinois in Gillespie for an insightful and constructive review. This work was supported by July 2007. To date, there have been no conﬁrmed cases of CCD in central U.S. Department of Agriculture Grants USDAG 2007–37610-18499 (to S. Rat- Illinois. All bees were immediately frozen on dry ice and kept at Ϫ80 °C until cliffe, G.E.R., and M.B.), USDAG 2008–3532-18831 (to M.R.B.), and USDA-NRI dissection. 2004–35604-14277 (to G.E.R.).

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